Alphaviruses are of general interest because they produce disease in a variety of animals, including humans, and because they replicate in invertebrates as well as vertebrates. Their broad replicative potential is essential for their transmission and thus for the spread of their associated diseases. Successful synthesis of many infectious progeny starts with the translation and copying of the infecting genomic into a complementary, or negative-strand, RNA template. Understanding the mechanism of alphavirus RNA synthesis and translation and of the regulation of negative strand transcription will contribute to our understanding of how cells function and regulate the expression of their genetic information. Furthermore, alphaviruses cause persistent infection in mosquitos and must have evolved a mechanism that allows viral replication but prevents Emg infected mosquitos which are their reservoir in nature. Therefore, our long range goal is to develop experimental methods that allow us to investigate the nature of the molecular mechanism by which alphaviruses, and most probably also rubella virus, create a viral replication complex and regulate their rate of transcription. Our three aims for the next granting period are: 1. To purify and characterize alphavirus replication complexes which will be isolated from cells infected with either parental virus, conditionally-lethal mutants, or defective-interfering particles containing DI RNA that is much shorter than genome RNA and to develop conditions that permit in vitro replication of exogeneous templates. 2. To identify host cell components that are associated with alphavirus replication complexes and determine their role in viral replication. 3. To define the functions of viral nonstructural proteins in negative strand synthesis and in the temporal cessation of negative strand synthesis. Using the infectious Sindbis clone, we will map the mutations in ts4 which prevent negative strand synthesis and in ts17 and ts133 which also possess the ability to synthesize negative strands late in infection, i.e., the 24R phenotype, and test the hypothesis that nsP2, in addition to nsP4, plays a role in the temporal cessation of negative strand synthesis. Using site-directed mutagenesis, we will determine which amino acid alterations in nsp4, other than the one in ts24 and 24R, allow the continuation of negative strand synthesis late in infection, i.e., give the 24R phenotype and we will define amino acids changes in nsPI, other than the one in ts11 , that makes negative strand synthesis temperature-sensitive and probe further the role of nsP 1 in negative strand synthesis.